Core A: PI: David L. Ayares, PhD Abstract: Precise genetic manipulation of the pig genome through a combination of somatic cell nuclear transfer and sophisticated gene modification tools offers the opportunity to provide an unlimited source of human-compatible donor organs, cells, and tissues for transplantation. As Core A, Revivicor will apply their advanced transgenic pig production platform to provide the GE source pig organs (kidney, heart, lung, and liver) necessary to meet the goals of the specific aims in Project 1 and Project 2. Elimination of immunogenic Gal?1,3Gal (Gal) sugars through inactivation of the ?1,3-galactosyl transferase gene (GTKO) was a critical first step, resulting in the elimination of hyperacute rejection (HAR), and prolonged survival of xenografts in non- human primate studies, compared to wild-type controls. To provide further protection protection from non-gal-mediated humoral rejection, next-generation GTKO pigs were produced with constitutive high-level expression of the complement regulatory genes, hCD46 and hCD55, individually and as triple transgenic pigs. In addition, for inhibition of coagulation and thrombosis, human anti-coagulant genes (EPCR, CD39, TFPI, and thrombomodulin), under endothelial cell-specific promoter systems, were added alone or in combination to produce donor pigs with up to six genetic modifications. The use of multi-cistronic ?2A? vectors has facilitated the production of these multi- transgenic pigs in which the added genes are not only co-expressed, but co-integrated in the genome, thus allowing propagation of new lines with reduced transgene segregation. Core A will utilize this multi-transgenic 2A pig platform to produce multiple combinations of six-gene (6GE) pigs that have already shown improved efficacy, and will further modify the source pig to inhibit T cell activation (expression of a dominant negative inhibitor of SLA class II (CIITA), and immunomodulation, through overexpression of the ?cloaking gene?, human CD47, to prevent activation of monocytes and other immune cells. In addition, towards prevention of spontaneous aggregation and sequestration of recipient platelets (and resulting thrombocytopenia in NHP), specific exons of porcine vWF will be replaced by their human equivalents using gene editing nuclease technologies. Core A will identify the optimal multi- gene mix, with an efficacious balance of gene expression levels, bioactivity, and synergistic effects, while maintaining a healthy herd, as needed to support the aims of Project 1 & 2.